1. The Field of the Invention
The invention relates generally to apparatus for analysis of biological samples which are subjected to rapid change of temperature. More specifically, the present invention relates to the direct, on-line analysis of DNA products derived from a thermal cycling apparatus.
2. The Background Art
The recent efforts to discover the genetic basis for human beings has raised the promise of cures for diseases heretofore incurable, the promise of extending the life of humans, and the promise of generally improving the quality of life for humans. The Human Genome Project being spearheaded in the United States is a major step toward realizing these promises which genetic engineering and its related technological areas have in store. Disadvantageously, discovering the entire DNA sequence of any mammal is a mammoth undertaking and the available technology is far behind the rate which is necessary if the Human Genome Project is to be completed in a reasonable period of time.
While alternative technologies have been proposed to simplify or speed up DNA sequencing, techniques using dideoxy reactions and gel electrophoresis still remain at the core of most efforts to improve DNA sequencing technology. Recent advances in gel-based methods have brought DNA sequencing technology to the rate of sequencing about one megabase per year but not to the megabase per day rates needed to complete the Human Genome Project in a reasonable period of time.
Even though techniques such as ultra-thin slab gel electrophoresis have improved to the point of carrying out electrophoresis separations at a desirable rate, (See Stegemann, J., Schwager, C., Erfle, H., Hewitt, N., Voss, H. Zimmerman, J. and Ansorge, W. (1991), Highspeed on-line DNA sequencing on ultrathin slab gels, Nucleic Acids Res. 19, 675-676; Kostichka, A. J., Marchbanks, M. L., Brumley, R. L., Jr., Drossman, H. and Smith, L. M. (1992), High speed automated DNA sequencing in ultrathin slab gels, Bio/Technology 10, 78-81) and multiple capillary electrophoresis devices have been built (Taylor, J. A. and Yeung, E. A. (1993), Multiplexed fluorescence detector for capillary electrophoresis using axial optical fiber illumination, Anal. Chem. 65, 956-960; Ueno, K. and Yeung, E. S. (1994), Simultaneous monitoring of DNA fragments separated by electrophoresis in a multiplexed array of 100 capillaries, Anal. Chem. 66, 1424-1431; Kambara, H. and Takahashi, S. (1993) Multiple-sheathflow capillary array DNA analyzer, Nature 361, 565-566; Takahashi, S., Murakami, K., Anazawa, T. and Kambara, H. (1994) Multiple sheath-flow gel capillary-array electrophoresis for multicolor fluorescent DNA detection, Anal. Chem. 66, 1021-1026; Huang, X. C., Quesada, M. A. and Mathies, R. A. (1992), Capillary array electrophoresis using laser-excited confocal fluorescence detection, Anal. Chem. 64, 967-972; Clark, S. M. and Mathies, R. A. (1993) High-speed parallel separation of DNA restriction fragments using capillary array electrophoresis, Anal. Biochem. 215, 163-170), the problem which has not been recognized in the art, or at least not solved, is that of feeding such electrophoresis instruments at the voracious rate at which they are capable of carrying out their procedures. It has not been recognized in the art that the front-end tasks of preparing samples for processing by these electrophoresis techniques have recently become the rate-limiting step in DNA analysis and particularly in DNA sequencing. Such front-end tasks include generation of libraries, ordering the large fragments and mapping and subcloning of smaller fragments. Other important front-end tasks also involve the preparation of templates, performance of reactions, purification of reaction products and loading of samples, before electrophoresis can begin.
It would be a significant advance in the art to provide a system which can carry out such front-end tasks quickly, efficiently, and accurately.
In particular, the front-end task of reliably and reproducibly subjecting relatively small DNA samples to thermal cycling has generally been an extremely time consuming step. Cyclic DNA amplification, using a thermostable DNA polymerase, allows automated amplification of specific DNA, widely known as the polymerase chain reaction or PCR. Automation of this process requires controlled and precise thermal cycling of reaction mixtures usually contained in a plurality of containers. In the past, the container of preference has been a standard, plastic microfuge tube.
Commercial programmable metal heat blocks have been used in the past to carry out the temperature cycling of samples in microfuge tubes through the desired temperature versus time profile. However, the inability to quickly and accurately adjust the temperature of the heater block through a large temperature range over a short time period, has rendered the use of the heater block type devices undesirable as a heat control system when carrying out the polymerase chain reaction.
Moreover, the microfuge tubes which are generally used have disadvantages. The material of the microfuge tubes, their wall thickness, and the geometry of the microfuge tubes is a hindrance to rapid heating and cooling of the sample contained therein. The plastic material and the thickness of the wall of microfuge tubes act as an insulator between the sample contained therein and the surrounding medium thus hindering transfer of thermal energy. Also, the geometry of the microfuge tube presents a small surface area to whatever medium is being used to transfer thermal energy. The continued use of microfuge tubes in the art, with their suboptimal geometry, indicates that the benefits of improved thermal transfer (which come by increasing the surface area of the sample container for samples of constant volume) has not been generally recognized in the art.
Furthermore, devices using water baths with fluidic switching (or mechanical transfer) have also been used as a thermal cycler for the polymerase chain reaction. Although water baths have been used in cycling a PCR mixture through a desired temperature versus time profile necessary for the reaction to take place, the high thermal mass of the water (and the low thermal conductivity of plastic microfuge tubes) has been significantly limiting as far as performance of the apparatus and the yields of the reaction are concerned.
Devices using water baths provide very slow thermal cycling performance and the yields of the reaction are less than desirable. This is because the water's thermal mass significantly restricts the maximum temperature versus time gradient which can be achieved thereby. Also, the water bath apparatus has been found to be very cumbersome due to the size and number of water carrying hoses and external temperature controlling devices for the water. Further, the need for excessive periodic maintenance and inspection of the water fittings for the purpose of detecting leaks in a water bath apparatus is tedious and time consuming. Finally, it is difficult with the water bath apparatus to control the temperature in the sample tubes with the desired accuracy.
U.S. Pat. No. 3,616,264 to Ray shows a thermal forced air apparatus for cycling air to heat or cool biological samples to a constant temperature. Although the Ray device is somewhat effective in maintaining a constant temperature within an air chamber, it does not address the need for rapidly adjusting the temperature in a cyclical manner according to a temperature versus time profile such as the polymerase chain reaction.
U.S. Pat. No. 4,420,679 to Howe and U.S. Pat. No. 4,286,456 to Sisti et al. both disclose gas chromatographic ovens. The devices disclosed in the Howe and Sisti et al. patents are suited for carrying out gas chromatography procedures but do not provide thermal cycling which is substantially any more rapid than that provided by any of the earlier described devices. Rapid thermal cycling, while potentially useful for many procedures, is particularly advantageous for carrying out the PCR. Devices such as those described in the Howe and Sisti et al. patents are not suitable for efficiently and rapidly carrying out such reactions.
Sample contamination also remains a significant problem for the user as well. When performing DNA amplification, minute contamination of DNA from another source can have disastrous consequences to the final results and conclusions of the procedure. One source of sample contamination comes from the process of the amplification of DNA of other samples to be run. Sample transfer techniques using pipettors or other means can contaminate the process. Likewise, even careful technicians can transfer DNA directly from the technician's body to the samples thereby reducing the confidence in the overall PCR process. As a consequence, sample preparation for DNA analysis is frequently carried out in "clean rooms" or at other clean locations within the facility which significantly increases the cost and space requirements for performing quality PCR.
It would also be a great advance in the art to provide a fully contained and automated system which would protect the user from being exposed to the DNA which is being amplified, as well as to protect the samples from cross-contamination. Using available technology, the user must handle samples following thermal cycling which may contain significantly higher concentrations of hazardous DNA species in order to subject the samples to further analysis. Thus, the exposure of the user to the amplified DNA is a serious problem in the state of the art.
Another disadvantage of the current state of the art is that the time to first result is very long. The amplification of DNA is only one front-end step in a series of analytical steps needed to arrive at the desired result. The current procedures can require eight hours or more of amplification and analysis before the result is known. This severely limits the use of the PCR technology for many application areas where answers are desired in minutes. DNA amplification with on-line analysis would be advantageous to the user. The labor intensive procedures required in the currently available devices are a hinderance to the efficient use of PCR technology.
U.S. Pat. Nos. 5,240,577 and 5,131,998 (Jorgenson) teach that enhanced 2-dimensional resolution can be obtained by the combination of liquid chromatography and capillary electrophoresis. However, the Jorgenson references do not suggest or teach that other steps necessary to DNA analysis, for example PCR reaction, should be combined in an automated on-line system.
In view of the forgoing, it would be an advance in the art to provide a system which can automate and expedite DNA analysis, including DNA sequencing, and particularly which can carry out front-end tasks such as cyclical DNA amplification and which does not require labor-intensive intervention by a technician.